Charge-dynamic polymers and delivery of anionic compounds

Field of the invention

The present invention relates to dynamic charge state cationic polymers that are useful for delivery of anionic molecules and methods using the polymers.

Background of the invention

The safe and efficient delivery of DNA to cells presents a formidable challenge and an obstacle to the clinical success of gene therapy. Anderson, W. F.

Human Gene Therapy. Nature, 392 Suppl. 25-30; Verma, I. M.; Somia, N.

Gene Therapy--Promises, Problems, and Prospects. Nature, 389, 239-242; Crystal, R. G.

Transfer of Genes to Humans: Early Lessons and Obstacles to Success. Science, 270, 404-410. Synthetic polymers have been investigated widely as gene delivery agents and are generally viewed as long-term alternatives to viruses due to their low immunogenicities and the ease with which they can be structurally modified. Luo, D.; Saltzman, W. M.

Synthetic DNA Delivery Systems. Nat. Biotechnol., 18, 33-37. Cationic polymers are particularly useful in this context because they form conjugates with negatively charged DNA, and the incorporation of new design elements into cationic polymers has resulted in advances toward functional gene delivery systems. Despite extensive work, however, polymers remain far less efficient than their viral counterparts.

For efficient gene transfer and expression to occur, a gene delivery agent (or vector) should overcome numerous intracellular barriers to transfection. Luo, D.; Saltzman, W. M.

Synthetic DNA Delivery Systems. Nat. Biotechnol., 18, 33-37. For example, a vector should be able to: 1) condense DNA into stabilized, nanometer-scale structures, 2) target cells and stimulate internalization, 3) prevent the degradation of DNA inside the cell, 4) target the cell nucleus, and 5) release DNA in the nucleus so that it is available for transcription. Progress has been made toward many of these barriers--cationic polymers are used to condense DNA into 50 to 200 nm particles (barrier 1), conjugation with cell-specific ligands can be used to target complexes and stimulate uptake (barrier 2), the incorporation of pH-buffering functionality into synthetic polymers provides protection against acidic intracellular environments (barrier 3), and the nuclear membrane has been "breached" (barrier 4). Kabanov, A. V.; Feigner, P. L.; Seymour, L. W., in Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998; Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R.

Polymer-Based Gene delivery with Low Cytotoxicity By a Unique Balance of Side-Chain Termini. Proc. Natl. Acad. Sci. USA, 98, 1200-1205; Midoux, P.; Monsigny, M.

Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes. Bioconjugate Chem., 10, 406-411; Boussif, O.; Lezoualc'H, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P.

A Versatile vector for Gene and Oligonucleotide Transfer Into Cells in Culture and In Vivo--Polyethyleneimine Proc. Natl. Acad. Sci. USA, 92, 7297-7301; Benns, J. M.; Choi, J.; Mahato, R. I.; Park, J.; Kim, S. W.

pH-sensitive Cationic Polymer Gene Delivery Vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) Comb Shaped Polymer. Bioconjugate Chem., 11, 67-645; Wolff, J. A.; Sebestyen, M. G.

Nuclear Security Breached. Nature Biotechnol., 19, 1118-1120; Rebuffat, A.; Bernasconi, A.; Ceppi, M.; Wehrli, H.; Verca, S. B.; Ibrahim, M.; Frey, B. M.; Frey, F. J.; Rusconi, S.

Selective Enhancement of Gene transfer by Steroid-Mediated Gene Delivery. Nature Biotechnol., 9, 1155-1161. These recent successes have fueled hopes of a "grand design" in which individual design elements could be assembled to create synthetic vectors that functionally mimic viruses. Wolff, J. A.

The "Grand" Problem of Synthetic Delivery. Nature Biotechnol., 20, 768-769. However, the breach of early barriers to transfection simply places increased significance on downstream barriers, and the design of materials to address the fifth and final barrier--the efficient and timely separation of polymer from DNA in the nucleus--has not been adequately addressed.

This "ultimate" barrier to efficient transfection presents a challenging problem from a design perspective, as designing methods to surmount it can introduce a functionality that is contrary to that required for efficient DNA condensation (i.e., barrier 1). Kircheis, R.; Wightman, L.; Wagner, E.

Design and Gene Delivery Activity of Modified Polyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358. Cationic polymers spontaneously self-assemble with anionic DNA through electrostatic interactions to form condensed interpolyelectrolyte complexes--a process that is driven entropically by the elimination of small salts (e.g., NaCl) formed upon complex formation. Kabanov, A. V.; Feigner, P. L.; Seymour, L. W., in Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998.

Cationic polymers undergo self-assembly with anionic plasmid DNA to form condensed complexes. The reverse of this process--the intracellular dissociation of DNA from condensed interpolyelectrolyte complexes--appears to be unfavorable under physiological conditions and presents a substantial obstacle to efficient gene delivery.

Although the effects of pH, temperature, salt concentration, and molecular weight on the dissociation of model interpolyelectrolyte complexes are generally well understood, the mechanisms through which dissociation occurs for polymer complexes in the cytoplasm or nucleus of a cell are currently unclear. Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V.

Recognition of DNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122, 8339-8343. That meaningful levels of transfection are observed using polymeric vectors suggests that dissociation does occur, presumably mediated by ion exchange with other intracellular polyelectrolytes. However, recent analytical experiments suggest that DNA/polycation complexes are stable toward intracellular dissociation and that the inefficiency of this "unpackaging" process presents a substantial physical barrier to transfection. Godbey, W. T.; Wu, K.; Mikos, A. G.

Tracking the Intracellular Path of Poly(ethyleneimine)/DNA Complexes for Gene Delivery. Proc. Natl. Acad. Sci. USA, 96, 5177-5181; Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A.

Vector Unpackaging as a Potential Barrier for Receptor-Mediated Polyplex Gene Delivery. Biotechnol. Bioeng., 67, 598-606.

Summary of the invention

The present invention provides a dynamic charge state cationic polymer, or more simply a polymer, that includes a polymeric backbone formed from monomeric units. One or more removable functional group is/are attached to the polymeric backbone. The dynamic charge state cationic polymer has a cationic charge density which is a characteristic of the polymeric backbone and the functional group attached to the polymeric backbone. The cationic charge density of the dynamic charge state cationic polymer decreases when the one or more of the removable functional group(s) is/are removed from the dynamic charge state cationic polymer. The present polymers can also be part of a copolymer where only one segment of the copolymer is the dynamic charge state cationic polymer. In some embodiments, the polymeric backbone comprises a polyamine, such as polyethyleneimine, polylysine, polyomithine or poly/lysine/ornithine. In some embodiments, the polymers contain side chains that have primary, secondary or tertiary amines. Other examples of suitable polymeric backbones include poly(propylene imine), poly(allyl amine), poly(vinyl amine), poly(amidoamine) (PAMAM), and dendrimers that are functionalized with terminal amine groups. Further examples include acrylate or methacrylate polymers such as poly(2-aminoethyl methacrylate), and the like. In some embodiments where amine functional groups are present in the polymer, primary amines may be functionalized either once or twice to provide a polymer that has a net negative charge once removal of the one or more removable functional group is complete. In the present polymers, the polymeric backbone can be linear, branched or hyperbranched, particularly when the backbone is polyethyleneimine.

In some embodiments, at least one of the one or more removable functional group(s) is a hydrolyzable group, such as a pendant ester. The one or more removable functional group(s) may also include a labile linkage, such as an ester, an anhydride, an orthoester, a phosphoester, an acetal, or an amide. In certain embodiments, the polymer has the formula:

##str00001##

When the polymer has the formula shown above, n may be an integer ranging from 5 to 100,000, x is an integer, and the mole percent of y ranges from 10 percent to 100 percent based on the total of x and y. In the present compounds, the identity of R is not particularly limited. For example, R can be an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or a heteroaryl group. R may also be carbon-containing, heteroatom-containing (N, S, O, P, etc.), linear, branched, an amino, an alkylamino, a dialkylamino, a trialkylamino, aryl, a heterocyclyl, a cyano, an amide, a carbamoyl, or the like. When the R group is alkyl, R can be methyl, ethyl, propyl, butyl, pentyl, hexyl, or combinations thereof.

In some embodiments, the polymers of the invention are biodegradable and biocompatible.

Other examples of polymers include compounds of the following formula:

##str00002##

In the above polymers, n may be an integer of from 5 to 100,000, A and B are linkers which may be the same or different and can be any substituted or unsubstituted, branched or unbranched chain of carbon atoms or heteroatoms; R.sub.1 may be a linker group or a covalent bond; X may be the same or different and can be a labile linkage, which, in some instances is negatively charged after cleavage; Y can be a linkage that is generally not as labile as X; R.sub.2 through R.sub.7 and R.sub.12 through R.sub.20 can have the value for R listed above and can be the same or different; R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are linkers or covalent bonds; and Z can be a covalent bond or a degradable linkage. In some embodiments X is an ester linkage and Y is an amide linkage, such as NR.sub.17. In some embodiments, R.sub.16 is NR.sub.21R.sub.22, R.sub.17 is H, and R.sub.18 is NR.sub.23R.sub.24. In some embodiments, R.sub.19 is NR.sub.25R.sub.26 and R.sub.20 is NR.sub.27R.sub.28. R.sub.21 through R.sub.28 can have the value for R listed above and can be the same or different. When Z is a covalent bond, the polymer backbone is non-degradable. The linkers A and B can be linkers that contain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.). Typically, these linkers are 1 to 30 atoms long, more preferably 1 to 15 atoms long. The linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, and alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. As would be appreciated by one of skill in this art, each of these groups may in turn be substituted. For some polymers the ester bond will generally be readily hydrolyzable whereas the amide bond is not readily hydrolyzable. This configuration allows more control over the change of cationic charge density of the polymer by altering the ratio of ester bonds and amide bonds present in the one or more removable functional group.

In the present polymers, the mole percent of the monomers comprising the polymeric backbone substituted with the one or more removable functional group range from 10 to 100 percent or from about 10 percent to about 100 percent. In additional embodiments, the mole percent of the monomers attached to the removable functional group may range from about 30 percent to 100 percent, 50 percent to 100 percent or 70 percent to 100 percent. The polymers of the present invention may have any desired molecular weight, such as from 1,000 to 100,000 grams/mole, or from about 2,000 to 50,000 grams/mole. The dynamic charge state cationic polymer can be associated with a ligand facilitating the delivery of the polymer to a specific target, such as a target cell. The present polymers can also be part of a copolymer, which can be composed of any other polymers, for example a polymer such as PEG or PEO which are commonly used to give stability toward protein adsorption. The present polymer is generally cationic, but different functional groups attached to the polymer can render the polymer zwitterionic. To impart a cationic charge to the polymer, the polymeric backbone or the attached functional groups can be positively charged. The present polymer may also be capable of buffering changes in pH which results from the make-up of the polymer backbone and/or the attached functional groups.

The present dynamic charge state cationic polymers may be non-immunogenic, non-toxic or both non-immunogenic and non-toxic. In the present polymers, the polymeric backbone can be degradable or nondegradable. The present polymers do not require that the degradation of the backbone occur at the same time as the shift in cationic charge. One skilled in the art will recognize that the measure of degradability will be commensurate with the environmental conditions and desired properties for any particular application for the present polymers. As one non-limiting example, for biomedical uses of the present polymers, the present invention contemplates polymers that degrade in a desired time frame (from an hour to a week to a month to a year) under physiological conditions typically found in the body or in a cell or cell compartment [e.g., pH ranges from about 5.0 (endosomal/lysosomal) to 7.4 (extracellular and cytosol), a temperature of about 37.degree. C. and an ionic strength of a typical physiological solution (generally around 130-150 mM NaCl, for example)]. In the present invention, the degradability of the polymer can be measured by a variety of methods, including, but not limited to, GPC (gel permeation chromatography).

The present invention also provides the present polymers complexed with one or more anionic molecules thereby forming an interpolyelectrolyte complex. Suitable anionic molecules may be naturally occurring, synthetic, or both. In some embodiments, suitable examples of anionic molecules include nucleic acids, such as RNA, DNA, and analogs thereof. In other embodiments, the anionic molecule is a synthetic polyanion. In still other embodiments, the polymers of the invention are complexed with an anionic molecule such as nucleic acids, such as RNA, DNA, or analogs thereof, and a synthetic polyanion. When the anionic molecule is a nucleic acid, the nucleic acid can have the sequence of a nucleic acid molecule of interest or its complement. As such, the nucleic acid can encode for a protein or a functional fragment thereof or be useful in antisense treatment or RNA interference. In some embodiments, the nucleic acid is a plasmid.

In other embodiments, the anionic molecule or agent may be a therapeutic molecule, diagnostic molecule, peptide, or carbohydrate, for example a macromolecular carbohydrate such as heparin.

The interpolyelectrolyte complex may have any desired size depending upon the intended use of the interpolyelectrolyte complex. For example, when the interpolyelectrolyte complex is used for nucleic acid delivery to a cell, the interpolyelectrolyte complex can be 50 nm to about 400 nm, or from about 50 to about 250 nanometers, in size. In other embodiments, the interpolyelectrolyte complex may be provided in a layered complex made up of one or more layers of the dynamic charge state cationic polymer and one or more layers of the anionic molecule.

In some embodiments, the present polymer or interpolyelectrolyte complex may be provided in a biologically compatible solution or a biological solution. Further, the polymer may be provided with a pharmaceutically acceptable excipient or another completely different polymer (e.g., another cationic polymer) which could be an "excipient" or could have an added function. Accordingly, the present compounds include pharmaceutical compositions that include any of the polymers or mixtures described herein.

The present invention also provides methods for delivering an anionic compound to a cell or tissue. The present methods involve contacting a composition that includes a present interpolyelectrolyte complex with a target cell thereby allowing the target cell to uptake the composition. The polymer of the present invention is designed such that when the interpolyelectrolyte complex enters the target cell, one or more of the removable functional group(s) is/are removed from the dynamic charge state cationic polymer which decreases the cationic charge density of the dynamic charge state cationic polymer. The decrease in the cationic charge density of the polymer may be caused by the introduction of anionic charges which promotes dissociation of the interpolyelectrolyte complex into the dynamic charge state cationic polymer and the anionic molecule allowing for delivery of the anionic molecule to the target cell or cell compartment, such as an endosome, cytosol or nucleus of the cell. In some methods, at least one of the one or more of the removable functional group(s) is removed from the dynamic charge state cationic polymer in a nucleus, endosome or cytosol of the target cell. In this manner, the interpolyelectrolyte complex may dissociate primarily in the desired compartment of the target cell and deliver the anionic molecule to the target cell compartment. The present methods may also involve providing the interpolyelectrolyte complex and/or preparing the interpolyelectrolyte complex. Generally, the interpolyelectrolyte complex will be prepared by mixing the dynamic charge state cationic polymer with the anionic molecule thereby allowing formation of the interpolyelectrolyte complex. In the methods where the anionic molecule is DNA, the DNA may be delivered to the nucleus of the cell so that it is stably incorporated into the genome of the target cell. In other embodiments, the DNA is not stably incorporated into the genome of the target cell.

In the present methods, the target cell or tissue can be in vitro or in vivo. Where the target cell or tissue is in vivo, the interpolyelectrolyte complex may be administered to a mammal. In some embodiments of the present methods, the cell is a eukaryotic cell.

In the present methods and polymers, removal of the one or more of the removable functional group(s) from the dynamic charge state cationic polymer may be at least partially hydrolytic, partially enzymatic and/or partially photolytic removal. The present polymers and methods may also be designed so that the removal of the one or more of the removable functional group(s) from the dynamic charge state cationic polymer occurs at a substantially constant rate or does not occur at a constant rate.

The present invention also provides kits containing the present polymers.

The invention also provides methods of preparing the polymers and methods of preparing microspheres and other pharmaceutical compositions containing the polymers.

In yet another aspect of the invention, the polymers are used to form nanometer-scale complexes with nucleic acids. The polynucleotide/polymer complexes may be formed by adding a solution of polynucleotide to a vortexing solution of the polymer at a desired DNA/polymer concentration. The weight to weight ratio of polynucleotide to polymer may range from 1:0.1 to 1:50, preferably from 1:1 to 1:20, more preferably from 1:1 to 1:10. The cationic polymers condense the polynucleotide into soluble particles typically 50-500 nm in size. These polynucleotide/polymer complexes may be used in the delivery of polynucleotides to cells. In some embodiments, these complexes are combined with pharmaceutical excipients to form pharmaceutical compositions suitable for delivery to animals including humans.

In another aspect of the invention, the polymers are used to encapsulate therapeutic, diagnostic, and/or prophylactic agents including polynucleotides to form microparticles. Typically these microparticles are one or more orders of magnitude larger than the polynucleotide/polymer complexes. The microparticles range from 1 micrometer to 500 micrometers. In some such embodiments, the microparticles allow for the delivery of labile small molecules, proteins, peptides, and/or polynucleotides to an individual. The microparticles may be prepared using any of the techniques known in the art to make microparticles, such as, for example, double emulsion and spray drying. In some embodiments, the microparticles may be used for pH-triggered delivery of the encapsulated contents due to the pH-responsive nature of the polymers (i.e., being more soluble at lower pH).

Brief description of the drawings

FIGS. 1-11 show the visualization of polycation/DNA interpolyelectrolyte complex formation and subsequent release of DNA from destabilized interpolyelectrolyte complexes of the present invention.

Detailed description of the invention

The present invention focuses on providing polymers that address the final physical barrier to efficient delivery--the timely intracellular dissociation of DNA from polymer/DNA interpolyelectrolyte complexes. The general approach of the present invention is based on the synthesis of cationic polymers that undergo dynamic changes in charge states (i.e., from cationic to "less cationic" or zwitterionic) to trigger the "unpackaging" of anionic molecules, such as DNA, from interpolyelectrolyte complexes. In one embodiment, the synthetic design is based on the introduction of side-chain esters to linear poly(ethylene imine) via conjugate addition chemistry. Without limiting the scope of the invention, it is believed that the dynamic introduction of carboxylate groups into these polymers, which occurs via the gradual hydrolysis of pendant ester groups, effectively lowers the cationic charge densities of the polymers and promotes the dissociation of polymer/anion complexes. The disclosed polymers undergo a shift in charged states as a function of time to initiate the efficient and timely unpackaging of DNA from condensed particles in the intracellular environment.

Affecting the timely intracellular dissociation of polycation/anionic complexes is an important and unsolved problem. The present polymers address this final physical barrier to anion delivery and can lead to effective new formulations for anion delivery and, importantly, allow existing solutions to earlier barriers to be exploited more fully. Prior research has generally approached this problem through the synthesis of hydrolytically degradable polycations or by the development of disulfide-crosslinked networks that use exposure to reductive intracellular environments to trigger degradation and enhance polymer dissociation. Peterson, H.; Merdan, T.; Kunath, K.; Fischer, D.; Kissel, T.

Poly(ethyleneimine-co-L-lactamide-co-succinamide): A Biodegradable Polyethyleneimine Derivative with an Advantageous pH-Dependent Hydrolytic Degradation for Gene Delivery. Bioconjugate Chem., 13, 812-821; Wang, J.; Mao, H.; Leong, K. W.

A Novel Biodegradable Gene Carrier Based on Polyphosphoester. J. Am. Chem. Soc., 123, 9480-9481; Lim, Y.; Kim, C.; Kim, K.; Kim, S. W.; Park, J.

Development of a Safe Gene Delivery System Using Biodegradable Polymer, Poly[.alpha.-(4-aminobutyl)-L-glycolic acid]. J. Am. Chem. Soc., 122, 6524-6525; Lynn, D. M.; Langer, R.

Degradable Poly(.beta.-Amino Esters): Synthesis, Characterization, and Self-Assembly with Plasmid DNA J. Am. Chem. Soc., 122, 10761-10768; Putnam, D.; Langer, R.

Poly(4-hydroxy-1-proline ester): Low-Temperature Polycondensation and Plasmid DNA Complexation. Macromolecules, 32, 3658-3662; Oupicky, D.; Parker, A. L.; Seymour, L. W.

Laterally Stabilized Complexes of DNA with Linear Reducible Polycations: Strategy for Triggered Intracellular Activation of DNA Delivery Vectors. J. Am. Chem. Soc., 124, 8-9; Pichon, C.; LeCam, E.; Guerin, B.; Coulaud, D.; Delain, E.; Midoux, P.

Poly[Lys-(AEDTP)]: A Cationic Polymer That Allows Dissociation of pDNA/Cationic Polymer Complexes in a Reductive Medium and Enhances Polyfection. Bioconjugate Chem., 13, 76-82; Gosselin, M. A.; Guo, W.; Lee, R. J.

Efficient Gene Transfer Using Reversibly Cross-Linked Low Molecular Weight Polyethyleneimine. Bioconjugate Chem., 12, 989-994. The present invention provides a different approach, based on synthetic polymers that shift charge states dynamically to trigger the "unpackaging" of DNA from interpolyelectrolyte complexes through the introduction of repulsive electrostatic interactions. One embodiment is outlined below:

##str00003##

The above scheme demonstrates the general concept of dynamic introduction of carboxylate groups to a cationic poly(amine) via side-chain ester hydrolysis. Assuming full protonation for illustrative purposes, the overall positive charge on polymer 1 is the sum of `n+m`, and this polymer is capable of forming interpolyelectrolyte complexes with polyanions such as DNA. Hydrolysis of the ester side chains in polymer 1 (assuming complete hydrolysis of all ester side chains) introduces `m` negative carboxylate groups and thus reduces the overall positive charge on polymer 2 to `n`, promoting dissociation of polymer complexes.

In this embodiment, the dynamic introduction of carboxylate groups into cationic polymers (via the gradual hydrolysis of pendant ester groups) effectively "neutralizes" or reduces the charge densities of these polymers and exerts a destabilizing influence that can promote the dissociation of polymer/DNA complexes. For example, the overall positive charge on polymer 1 is reduced from (n+m)+ to (n)+ upon hydrolysis (above scheme, assuming full protonation for illustrative purposes and complete hydrolysis of ester groups in polymer 2). One skilled in the art will recognize that this charge shifting capability can be readily applied to other polymers.

In some embodiments, the present polymers are based on linear poly(ethylene imine) (PEI) as the structural template from which polymers are synthesized. Linear PEI is an attractive template for several reasons: 1) it is commercially available or can be synthesized directly via the ring-opening polymerization of substituted oxazolines, 2) it is a proven gene transfer agent already capable of surmounting many "early" barriers to transfection, and 3) the linear array of secondary amines facilitates the introduction of new functionality through well defined chemistry. Odian, G., Principles of Polymerization, John Wiley and Sons, Inc., New York, 1991; Kircheis, R.; Wightman, L.; Wagner, E.

Design and Gene Delivery Activity of Modified Polyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358; Yin, R.; Zhu, Y.; Tomalia, D. A.

Architectural Copolymers: Rod-Shaped, Cylindrical Dendrimers. J. Am. Chem. Soc., 120, 2678-2679. The methods below can also be applied to branched or hyperbranched PEI (also an effective gene transfer agent). Boussif, O.; Lezoualc'H, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P.

A Versatile Vector for Gene and Oligonucleotide Transfer Into Cells in Culture and In Vivo--Polyethyleneimine Proc. Natl. Acad. Sci. USA, 92, 7297-7301; Kircheis, R.; Wightman, L.; Wagner, E.

Design and Gene Delivery Activity of Modified Polyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358. In some embodiments, the present invention modifies polymers that are already known as effective polyanion delivery agents.

In some embodiments, the synthetic strategy is based on the conjugate addition of the secondary amines of PEI to acrylate compounds. The exhaustive functionalization of linear PEI has been demonstrated in the context of dendrimer synthesis using methyl acrylate, yielding ester-functionalized polymer 1 (R=methyl, n=0) having regularly repeating tertiary amines in the polymer backbone. Yin, R.; Zhu, Y.; Tomalia, D. A.

Architectural Copolymers: Rod-Shaped, Cylindrical Dendrimers. J. Am. Chem. Soc., 120, 2678-2679.

The introduction of the alkyl ester groups in some of the present polymers may influence the pKa's and increase the steric bulk surrounding the amines, and may consequently affect the ability of the polymers to form complexes with DNA. To investigate the relationships between charge density, backbone substitution, and interpolyelectrolyte complex formation (methods described below) a family of polymers with a range of mole percent substitution (i.e., from about 10% to 100% functionalized) have been synthesized. Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R.

Polymer-Based Gene Delivery with Low Cytotoxicity by a Unique Balance of Side-Chain Termini. Proc. Natl. Acad. Sci. USA, 98, 1200-1205; Jeong, J. H.; Song, S. H.; Lim, D. W.; Lee, H.; Park, T. G.

DNA Transfection Using Linear Poly(ethyleneimine) Prepared by Controlled Acid Hydrolysis of Poly(2-ethyl-2-oxazoline). J. Control. Release, 73, 391-399. Similarly, polymers with varying hydrophobicities and charge densities have been synthesized by conjugate addition to more hydrophobic acrylates (i.e., where R=ethyl, propyl, butyl, or combinations thereof). Because both transfection and the "unpackaging" of DNA from interpolyelectrolyte complexes appear to be related to polycation molecular weight (with shorter polymers tending to dissociate more readily than larger polymers), individual syntheses were conducted using PEI of different molecular weights and polydispersities to explore these relationships and optimize polymer behavior. Godbey, W. T.; Wu, K. K.; Mikos, A. G.

Size Matters: Molecular Weight Affects the Efficiency of Poly(ethyleneimine) as a Gene Delivery Vehicle. J. Biomed. Mater. Res., 45, 268-275; Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A.

Vector Unpackaging as a Potential Barrier for Receptor-Mediated Polyplex Gene Delivery. Biotechnol. Bioeng., 67, 598-606.

As described above, polycations and plasmid DNA spontaneously self-assemble under physiological conditions to form nanometer-scale interpolyelectrolyte complexes. These complexes may be destabilized by exposure to high salt concentrations or by the presence of other polyelectrolytes that initiate polyion exchange processes. Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V.

Recognition of DNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122, 8339-8343. Agarose gel electrophoresis provides for convenient visualization of polycation/DNA interpolyelectrolyte complex formation and is used as a qualitative measure for the subsequent release of DNA from destabilized complexes. Lynn, D. M.; Langer, R.

Degradable Poly(.beta.-Amino Esters): Synthesis, Characterization, and Self-Assembly with Plasmid DNA J. Am. Chem. Soc., 122, 10761-10768; Putnam, D.; Langer, R.

Poly(4-hydroxy-1-proline ester): Low-Temperature Polycondensation and Plasmid DNA Complexation. Macromolecules, 32, 3658-3662; Wang, J.; Mao, H.; Leong, K. W.

A Novel Biodegradable Gene Carrier Based on Polyphosphoester. J. Am. Chem. Soc., 123, 9480-9481. To obtain more quantitative information about the biophysical interactions between DNA and the polymers synthesized above, one may also use an established fluorescence-based ethidium bromide exclusion assay to investigate the kinetics of complex destabilization. Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V.

Recognition of DNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122, 8339-8343.

The kinetics of ester hydrolysis and interpolyelectrolyte complex disruption of some of the present polymers was evaluated over a range of pH, temperature, and salt concentration, focusing on those conditions likely to be encountered by these materials during transfection (e.g., 37.degree. C., 150 mM NaCl, and pH values ranging from 5.1 to 7.2 to approximate the pH within endosomal vesicles and the cytoplasm, respectively). Interpolyelectrolyte complexes were formed at various polymer/DNA ratios by standard mixing protocols. Dynamic light scattering (DLS) was used to determine the size distributions of the complexes and the relationships between backbone substitution, cation charge density, side-chain hydrolysis, and interpolyelectrolyte complex particle charge. Zeta-potential analysis may also be used to characterize the polymer/DNA complexes. Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V.

Recognition of DNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122, 8339-8343; Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R.

Polymer-Based Gene Delivery with Low Cytotoxicity by a Unique Balance of Side-Chain Termini. Proc. Natl. Acad. Sci. USA, 98, 1200-1205; Lynn, D. M.; Langer, R.

Degradable Poly(.beta.-Amino Esters): Synthesis, Characterization, and Self-Assembly with Plasmid DNA J. Am. Chem. Soc., 122, 10761-10768; Gonzalez, H.; Hwang, S. J.; Davis, M. E.

New Class of Polymers for the Delivery of Macromolecular Therapeutics. Bioconjugate Chem., 10, 1068-1074.

The present polymers have been rationally designed to promote the intracellular dissociation of DNA from interpolyelectrolyte complexes. The functional polymers may change charge states as a function of time, allowing the initial formation of polymer DNA complexes (to address early barriers to transfection) and facilitating intracellular dissociation through the introduction of repulsive electrostatic interactions (to address late barriers). An appropriate balance of charge density and side-chain hydrolysis allows these competing factors to be addressed on a time scale relevant to biological anion delivery/gene transfer and expression. The present polymers may also be designed to have a desired rate of hydrolysis so that the charge shift of the polymer occurs on the desired time scale.

The polymers may be used in the pharmaceutical/drug delivery arts to deliver polynucleotides, proteins, small molecules, peptides, antigen, drugs, or the like to a patient, tissue, organ, cell, or the like.

The polymers may also be used to complex polynucleotides and thereby enhance the delivery of polynucleotide and prevent their degradation. The polymers may also be used in the formation of nanoparticles or microparticles containing encapsulated agents. Due to some of the polymers' properties of being biocompatible and biodegradable, these formed particles are also biodegradable and biocompatible and may be used to provide controlled, sustained release of the encapsulated agent. These particles may also be responsive to pH changes.

Polymers

The present polymers are dynamic charge state cationic polymers that have a cationic charge density which is a characteristic of the polymeric backbone and the functional groups attached to the polymeric backbone. The polymers are designed such that the cationic charge density of the dynamic charge state cationic polymer decreases when one or more of the removable functional group(s) is removed from the dynamic charge state cationic polymer.

Based on these criteria, the polymer backbone is not particularly limited. In some embodiments, the polymer backbone is positively charged, whereas in others the polymeric backbone is neutral. Generally speaking, the charge of the polymer backbone is measured under physiological conditions, such as at physiological pH. In some instances the polymer backbone is made up of repeating units of polyamines, such as polyethyleneimine, polylysine, polyornithine or poly/lysine/ornithine, because such polymers provide the desired cationic charge density and are easy to manipulate. Specific examples of such suitable polymeric backbones include poly(propylene imine), poly(allyl amine), poly(vinyl amine), poly(amidoamine) (PAMAM), and dendrimers that are functionalized with terminal amine groups. Further examples of polymeric backbones suitable for use in the present invention include acrylate or methacrylate polymers such as poly(2-aminoethyl methacrylate), and the like. In some embodiments, where amine functional groups are present in the polymer, primary amines may be functionalized either once or twice to provide a polymer that has a net negative charge once removal of the one or more removable functional group(s) is complete. In the present polymers, the polymeric backbone may be linear, branched or hyperbranched.

The present polymer is generally cationic, but different functional groups attached to the polymer can render the polymer zwitterionic. The present polymer may also be capable of buffering changes in pH which results from the make-up of the polymer backbone and/or the attached functional groups.

Similar to the backbone, the identity of the one or more removable functional group(s) of the present polymers is not particularly limited as long as removal of the one or more removable functional group(s) decreases the cationic charge density of the polymer. As used herein, "removable functional group" means a chemical group that, upon removal, will decrease the cationic charge density of the polymer. As will be apparent to the skilled artisan, polymers whose cationic charge density decreases in this manner can have a variety of features. For example, the removable functional group may be positively charged so that removal of the removable functional group reduces cationic charge density. This may be particularly important where the polymeric backbone is not positively charged. In other embodiments the removable functional group may be positively charged or neutral prior to removal, but provide a negatively charged species after it is removed from the polymer. One example of such a scheme is provided when the removable group contains a hydrolyzable ester. Other configurations that achieve the charge shifting properties of the present polymers will be apparent to those skilled in the art. When removable functional groups provide a negatively charged species after removal from the polymer backbone and the backbone itself is neutral, then the present polymers can shift from being cationic to anionic when the removable functional group is removed.

Examples of removable functional groups suitable for use in the present polymers include side chains that have primary, secondary or tertiary amines. Primary amines useful in the present polymers include, but are not limited to, methylamine, ethylamine, isopropylamine, aniline, substituted anilines, and ethanolamine. In some embodiments, at least one of the one or more removable functional group(s) is a hydrolyzable group, such as a pendant ester. Specific examples for the one or more removable functional group(s) may also include a labile linkage, such as an ester, an anhydride, an orthoester, a phosphoester, an acetal, or an amide.

The present polymer is generally cationic, but different functional groups attached to the polymer may render the polymer zwitterionic. The present polymer may, also be capable of buffering changes in pH which results from the make-up of the polymer backbone and/or the attached functional groups.

More specifically, polymers having the following structure are suitable for use in the present invention:

##str00004##

The description continues in the full USPTO document.